Drug combination

ABSTRACT

The invention provides a composition which comprises (a) a PDE3/PDE4 inhibitor which is 9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one or a pharmaceutically acceptable acid addition salt thereof and (b) a β 2 -adrenergic receptor agonist.

This application claims priority from U.S. provisional patentapplication No. 61/799,177 filed 15 Mar. 2013, which is incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to a new combination of drugs which hassurprising therapeutic efficacy in the treatment of respiratory andinflammatory disorders.

BACKGROUND OF THE INVENTION

There are a number of different therapeutic approaches to treatingrespiratory diseases such as asthma and chronic obstructive pulmonarydisease (COPD). For example, corticosteroids, β₂-adrenergic receptoragonists, phosphodiesterase (PDE) 4 inhibitors, PDE 3 inhibitors,leukotriene receptor antagonists, epidermal growth factor receptor(egfr) kinase inhibitors, p38 kinase inhibitors, NK1 agonists andmuscarinic receptor antagonists are all known for use in the treatmentof respiratory diseases.

RPL554(9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one)is a dual PDE3/PDE4 inhibitor and is described in WO 00/58308. As acombined PDE3/PDE4 inhibitor, RPL554 has both anti-inflammatory andbronchodilatory activity and is useful in the treatment of respiratorydisorders such as asthma and chronic obstructive pulmonary disease(COPD).

It is known that different classes of respiratory drugs may be used incombination for the treatment of respiratory diseases. However,synergistic interaction between the components of such combinations israrely observed.

SUMMARY OF THE INVENTION

It is a surprising finding of the present invention that RPL554 iscapable of potentiating the activity of β₂-adrenergic receptor agonists.RPL554 and β₂-adrenergic receptor agonists therefore interactsynergistically in combination to provide an improved therapeuticeffect.

True synergistic interactions between drugs are rare. The presence of asynergistic interaction can be determined by, for example, the Berenbaummethod, the Bliss Independence (BI) criterion and/or the LoeweAdditivity (LA) model through curved isoboles (see Berenbaum, 1977;Greco et al., 1995; Grabovsky and Tallarida, 2004; Tallarida, 2006;Goldoni and Johansson, 2007; Tallarida and Raffa, 2010).

According to the Berenbaum method, synergy for a combination is detectedby first determining dose-response curves for each of the constituentdrugs as monotherapies in order to identify a low and high dose of eachdrug. The effect of a combination of the low doses of each drug is thenmeasured. If a combination of the low doses of each drug produces agreater response than either high dose alone as monotherapy, then thereis true synergy between the two drugs.

The present inventors have surprisingly found that a true synergisticeffect according to the Berenbaum method arises when RPL554 is combinedwith a β₂-adrenergic receptor agonist. The enhanced therapeutic effectthat is obtained whilst using low doses of each constituent drug ishighly desirable in a clinical context, and for example reduces the sideeffects experienced by the patient.

Accordingly, the present invention provides a composition whichcomprises (a) a PDE3/PDE4 inhibitor which is9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-oneor a pharmaceutically acceptable acid addition salt thereof and (b) aβ₂-adrenergic receptor agonist.

The invention also provides a pharmaceutical composition comprising acomposition according to the invention and one or more pharmaceuticallyacceptable carriers, diluents, or excipients

The invention also provides a method of treating a disease or conditionwhich is based on (i) acute or chronic obstruction of vessels or bronchior (ii) acute or chronic inflammation, in a subject in need thereof,which method comprises administering to said subject (a) a PDE3/PDE4inhibitor as defined herein and (b) a β₂-adrenergic receptor agonist.

The invention also provides a product comprising (a) a PDE3/PDE4inhibitor as defined herein and (b) a β₂-adrenergic receptor agonist forsimultaneous, separate or sequential use in the treatment of a diseaseor condition as defined herein.

The invention also provides use of (a) a PDE3/PDE4 inhibitor as definedherein in the manufacture of a medicament for simultaneous, separate orsequential use in the treatment of a disease or condition as definedherein in combination with (b) a β₂-adrenergic receptor agonist.

The invention also provides use of (b) a β₂-adrenergic receptor agonistin the manufacture of a medicament for simultaneous, separate orsequential use in the treatment of a disease or condition as definedherein in combination with (a) a PDE3/PDE4 inhibitor as defined herein.

The invention also provides use of (a) a PDE3/PDE4 inhibitor as definedherein and (b) a β₂-adrenergic receptor agonist in the manufacture of amedicament for use in the treatment of a disease or condition as definedherein.

The invention also provides a composition of the invention for use inthe treatment of a disease or condition as defined herein.

The invention also provides a PDE3/PDE4 inhibitor as defined herein foruse in the treatment of a disease or condition as defined herein incombination with a β₂-adrenergic receptor agonist.

The invention also provides a β₂-adrenergic receptor agonist for use inthe treatment of a disease or condition as defined herein in combinationwith a PDE3/PDE4 inhibitor as defined herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Line graph representing inhibition of contraction of humanisolated bronchial preparations to EFS following 50 min incubation withRPL554. Points shown are from experiments performed with samples of n=5different subjects and they are represented as mean±SEM; ***P<0.001 vscontrol.

FIG. 2: Human bronchial relaxation of RPL554 and salbutamol onsub-maximal contraction by acetylcholine. Points shown are fromexperiments performed with samples of n=5 different subjects and theyare represented as mean±SEM; ***P<0.001 vs salbutamol.

FIG. 3: Effect of increasing dose of RPL554 on contractile effect ofhistamine in passively sensitized human isolated bronchi. Points shownare from experiments performed with samples of n=5 different subjectsand they are represented as mean±SEM ***P<0.001 vs passively sensitizedcontrol.

FIG. 4: Low concentrations interaction (10 nM and 100 nM) betweensalbutamol and RPL554. Data are from experiments performed with samplesof n=5 different subjects and they are represented as mean±SEM. *P<0.05and **P<0.01.

FIG. 5: Interaction surfaces obtained from response surface analysis ofBI drug interaction model for the combination of RPL554 plus salbutamol.The horizontal-axis indicates the concentration of compounds and thevertical-axis represent the ΔE (relaxation, %). The 0-plane indicates BIinteractions whereas the volume above the 0-plane represents synergistic(positive ΔE) interactions. The magnitude of interactions is directlyrelated to ΔE and the different tones in the 3D plots representdifferent percentile bands of synergy (10%). Each point intersectionrepresents the mean of experiments performed on samples from differentsubjects (n=5).

FIG. 6: Bar graph representing the relaxation response to salbutamol(open column; SALB, nM), RPL554 (closed column; nM) the additiveresponse of each dose combination (stippled column; Additive) and theobserved relaxation response for each dose combination (dark stippledcolumn; Combination 1:1)). The concentrations of each agonist are shownon the X axis. Each bar represents the mean and vertical lines representthe standard deviation (N=5). In the case of the additive response, theSD was estimated using the methods of Tallarida and Raffa (2010). *P<0.05 (adjusted) of additive response using a one sample t-test.

FIG. 7: Reduction in airways obstruction (induced by the intravenous(iv.) administration of bombesin (2 μg/ml; 5 ml/hr)) following the iv.administration of RPL554 alone (•) or in combination with 20 μg/kgsalbutamol (▪).

FIG. 8: Reduction in mean arterial blood pressure following the iv.administration of RPL554 alone (•) or in combination with 20 μg/kgsalbutamol (▪).

FIG. 9: Reduction in airways obstruction (induced by the intravenous(iv.) administration of bombesin (2 μg/ml; 5 ml/hr)) following the iv.administration of salbutamol.

FIG. 10: Reduction in mean arterial blood pressure following the iv.administration of salbutamol.

DETAILED DESCRIPTION OF THE INVENTION

The following abbreviations are used herein:

RPL554:9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one;ANOVA: analysis of variance; BI: Bliss Independence; COX:cyclooxygenase; EC30: concentration required to cause a 30% maximaleffect; EC50: concentration required to cause a 50% maximal effect;EC70: concentration required to cause a 70% maximal effect; EFS:electrical field stimulation; Emax: maximal effect; KH: Krebs-Henseleitbuffer solution; LA: Loewe Additivity; and PDE: phosphodiesterase.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise undesirable. For example, the term“pharmaceutically acceptable carrier” refers to a material that can beincorporated into a composition and administered to a subject/patientwithout causing undesirable biological effects or interacting in adeleterious manner with other components of the composition. Suchpharmaceutically acceptable materials typically have met the requiredstandards of toxicological and manufacturing testing, and include thosematerials identified as suitable inactive ingredients by the U.S. Foodand Drug administration.

The term “pharmaceutically acceptable acid addition salt” refers to anacid addition salt of a pharmaceutical which is not biologically orotherwise undesirable. Such pharmaceutically acceptable acid additionsalts are well known to the skilled person.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a subject in need of treatment.In particular, an “effective” amount is that amount needed to obtain thedesired result, and a “therapeutically effective” amount is that amountneeded to obtain the desired therapeutic effect. For example, foragonizing a β₂-adrenergic receptor, an “effective amount” is aβ₂-adrenergic receptor-agonizing amount. Similarly, a therapeuticallyeffective amount for treating chronic obstructive pulmonary disease(COPD) is that amount that will achieve the desired therapeutic result,which may be disease prevention, amelioration, suppression oralleviation.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as COPD) in a subject,such as a mammal (particularly a human) that includes: (a) preventingthe disease or medical condition from occurring, i.e., prophylactictreatment of a subject; (b) ameliorating the disease or medicalcondition, i.e., eliminating or causing regression of the disease ormedical condition in a subject; (c) suppressing the disease or medicalcondition, i.e., slowing or arresting the development of the disease ormedical condition in a subject; or (d) alleviating the symptoms of thedisease or medical condition in a subject. For example, the term“treating COPD” would include preventing COPD from occurring,ameliorating COPD, suppressing COPD, and alleviating the symptoms ofCOPD. The term “subject” is intended to include those animals, such ashumans, that are in need of treatment or disease prevention, that arepresently being treated for disease prevention or treatment of aspecific disease or medical condition, as well as test subjects in whichcompositions of the invention are being evaluated or being used in anassay, for example an animal model.

The PDE3/PDE4 Inhibitor

The PDE3/PDE4 inhibitor used in the present invention is9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one(also known as RPL554) or a pharmaceutically acceptable acid additionsalt thereof.

Typically, the PDE3/PDE4 inhibitor is9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one.Thus the free base of9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-oneis preferred.

β₂-Adrenergic Receptor Agonist

A β₂-adrenergic receptor agonist is a compound that acts onβ₂-adrenergic receptors, thereby causing smooth muscle relaxation. Askilled person can determine whether a given compound is a β₂-adrenergicreceptor agonist without difficulty using assays well known to thoseskilled in the art.

β₂-adrenergic receptor agonists are well known to act similarly as aclass of drugs despite structural differences between the compounds inthis class. It is a finding of the present invention that RPL554 iscapable of potentiating the activity of salbutamol. In view of thesimilar behaviour of all members of the β₂-adrenergic receptor drugclass, it follows that a synergistic interaction with RPL554 can beexpected for all compounds with β₂-adrenergic receptor agonist activity.

Examples of β₂-adrenergic receptor agonists β₂-adrenoreceptor agonists)include albuterol, bitolterol, fenoterol, formoterol, indacaterol,isoetharine, levalbuterol, metaproterenol, pirbuterol, salbutamol,salmefamol, salmeterol, terbutaline, and the like. Other examples ofβ₂-adrenergic receptor agonists include3-(4-{[6-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-phenyl]ethyl}amino)hexyl]oxy}butyl)benzenesulfonamideand3-(-3-{[7-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino)heptyl]oxy}-propyl)benzenesulfonamideand related compounds disclosed in WO 02/066422 (Glaxo Group Ltd.);3-[3-(4-{[6-([(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino)hexyl]oxy}butyl)phenyl]imidazolidine-2,4-dioneand related compounds disclosed in WO 02/070490 (Glaxo Group Ltd.);3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)-hexyl]oxy}butyl)benzenesulfonamide,3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)-hexyl]oxy}butyl)-benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamideand related compounds disclosed in WO 02/076933 (Glaxo Group Ltd.);4-{(1R)-2-[(6-{2-[(2,6-dichlorobenzyl)oxy]ethoxy}hexyl)amino]-1-hydroxyethyl}-2-(hydroxymethyl)phenoland related compounds disclosed in WO 03/024439 (Glaxo Group Ltd.);N-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(-3-formamido-4-hydroxyphenyl)ethylamineand related compounds disclosed in U.S. Pat. No. 6,576,793 to Moran etal.;N-{2-[4-(3-phenyl-4-methoxyphenyl)aminophenyl]ethyl}-(R)-2-hydroxy-2-(8-hydroxy-2(1H)-quinolinon-5-yl)ethylamineand related compounds disclosed in U.S. Pat. No. 6,653,323 to Moran etal. The β₂-adrenoreceptor agonist may be a crystalline monohydrochloridesalt ofN-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamine.Typically, the β₂-adrenergic receptor agonist will be administered in anamount sufficient to provide from about 0.05-500 μg per dose.

Typically, the β₂-adrenergic receptor agonist is salbutamol, albuterol,bitolterol, fenoterol, formoterol, indacaterol, isoetharine,levalbuterol, metaproterenol, pirbuterol, salmefamol, salmeterol orterbutaline.

Preferably, the β₂-adrenergic receptor agonist is salbutamol,salmeterol, formoterol, albuterol or pirbuterol.

Most preferably the β₂-adrenergic receptor agonist is salbutamol.

The β₂-adrenergic receptor agonists are optionally in the form of theirracemates, their enantiomers, their diastereomers, and mixtures thereof,and optionally their pharmaceutically acceptable acid addition salts.Typical examples of suitable acids for the formation of addition saltsof the β₂-adrenergic receptor agonists are hydrochloric acid,hydrobromic acid, sulphuric acid, phosphoric acid, methanosulphonicacid, acedic acid, fumaric acid, succinic acid, maleic acid, andtrifluoroacetic acid. Furthermore, mixtures of the aforementioned saltscan be used.

Compositions, Combinations, Pharmaceutical Compositions and Formulations

The compositions of the invention comprise (a) a PDE3/PDE4 inhibitorwhich is9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-oneor a pharmaceutically acceptable acid addition salt thereof and (b) aβ₂-adrenergic receptor agonist.

Typically, the composition of the invention is a fixed combination. In afixed combination, the PDE3/PDE4 inhibitor and the β₂-adrenergicreceptor agonist are present in the same composition. The fixedcombination can be used for simultaneous administration of the PDE3/PDE4inhibitor and the β₂-adrenergic receptor agonist. Typically, the fixedcombination is a dry powder composition (which is preferably suitablefor delivery from a dry powder inhaler), a solution which is suitablefor delivery from a nebulizer, or a solution or suspension which issuitable for delivery from a pressurised metered dose inhaler.

Thus, for example, the fixed combination is preferably a dry powdercomposition comprising both the PDE3/PDE4 inhibitor and theβ₂-adrenergic receptor agonist. Alternatively, the fixed combination canbe a solution, typically an aqueous solution, comprising both thePDE3/PDE4 inhibitor and the β₂-adrenergic receptor agonist, which issuitable for delivery from a nebulizer. Alternatively, the fixedcombination can be a solution or suspension comprising both thePDE3/PDE4 inhibitor and the β₂-adrenergic receptor agonist, which issuitable for delivery from a pressurised metered dose inhaler.

The two components in a fixed combination are typically intermixed.

Alternatively, the composition of the invention may be a freecombination. In a free combination, the active components (a) and (b)are typically separate from each other and packaged in one unit forsimultaneous, substantially simultaneous, separate or sequentialadministration.

Typically, the composition is a pharmaceutical composition which furthercomprises one or more pharmaceutically acceptable carriers, diluents, orexcipients in addition to the PDE3/PDE4 inhibitor and the β₂-adrenergicreceptor agonist. The compositions may contain other therapeutic and/orformulating agents if desired. A preferred example of anothertherapeutic agent is a muscarinic receptor antagonist. Examples ofmuscarinic receptor antagonists include atropine, hyoscine,glycopyrrolate (glycopyrronium), ipratropium, tiotropium, oxitropium,pirenzepine, telenzepine, aclidinium and umeclidinium. Preferredexamples of muscarinic receptor antagonists include atropine,glycopyrronium, ipratropium bromide or tiotropium bromide.

Compositions of the present invention are typically administered to asubject in the form of a pharmaceutical composition. Such pharmaceuticalcompositions may be administered to the subject by any acceptable routeof administration including, but not limited to, inhaled, oral, nasal,topical (including transdermal) and parenteral modes of administration.Administration by inhalation is preferred. Further, the compositions ofthe invention may be administered, for example orally, in multiple dosesper day, in a single daily dose or a single weekly dose. It will beunderstood that any form of the active agents used in the composition ofthe invention, (i.e. free base, pharmaceutically acceptable salt,solvate, etc.) that is suitable for the particular mode ofadministration can be used in the pharmaceutical compositions discussedherein.

The pharmaceutical compositions of this invention typically contain atherapeutically effective amount of an active agent. Those skilled inthe art will recognize, however, that a pharmaceutical composition maycontain more than a therapeutically effective amount, i.e., bulkcompositions, or less than a therapeutically effective amount, i.e.,individual unit doses designed for multiple administration to achieve atherapeutically effective amount. In one embodiment, the compositionwill contain from about 0.01-95 wt % of active agent, including, fromabout 0.01-30 wt %, such as from about 0.01-10 wt %, with the actualamount depending upon the formulation itself, the route ofadministration, the frequency of dosing, and so forth. In anotherembodiment, a composition suitable for inhalation, for example,comprises from about 0.01-30 wt % or active agent with yet anotherembodiment comprises from about 0.01-10 wt % active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular subject or typeof medical condition or disease state. In this regard, the preparationof a suitable composition for a particular mode of administration iswell within the scope of those skilled in the pharmaceutical arts.Additionally, carriers or excipients used in such compositions arecommercially available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; compressed propellant gases, such aschlorofluorocarbons and hydrofluorocarbons; and other non-toxiccompatible substances employed in pharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent/active ingredient with apharmaceutically acceptable carrier and one or more optionalingredients. The resulting uniformly blended mixture may then be shapedor loaded into tablets, capsules, pills, canisters, cartridges,dispensers and the like using conventional procedures and equipment.

Typically, the pharmaceutical compositions are suitable for inhaledadministration. The pharmaceutical composition may be for administrationby dry powder inhaler (DPI) or metered-dose inhaler (MDI).

Suitable compositions for inhaled administration will typically be inthe form of an aerosol or a powder, for instance a dry powdercomposition. Such compositions are generally administered usingwell-known delivery devices, such as a nebulizer inhaler, a dry powderinhaler, or a metered-dose inhaler, examples of which are describedbelow.

Alternatively, a composition comprising the active agent(s)/activeingredient(s) may be administered by inhalation using a nebulizerinhaler. Such nebulizer devices typically produce a stream of highvelocity air that causes the composition to spray as a mist that iscarried into a subject's respiratory tract. Accordingly, when formulatedfor use in a nebulizer inhaler, the active agent(s)/active ingredient(s)is typically dissolved in a suitable carrier to form a solution.Alternatively, the active agent(s)/active ingredient(s) can bemicronized and combined with a suitable carrier to form a suspension ofmicronized particles of respirable size, where micronized is typicallydefined as having particles in which at least about 90 percent of theparticles have a mass median diameter of less than about 10 μm. The term“mass median diameter” means the diameter such that half the mass of theparticles is contained in particles with larger diameter and half iscontained in particles with smaller diameter.

Suitable nebulizer devices include the Respimat® Soft Mist™ Inhaler(Boehringer Ingelheim), the AERx® Pulmonary Delivery System (AradigmCorp.), and the PARI LC Plus Reusable Nebulizer (Pari GmbH). Anexemplary composition for use in a nebulizer inhaler comprises anisotonic aqueous solution comprising from about 0.05 μg/mL to about 10mg/mL of a RPL554. In one embodiment, such a solution has a pH of about3.5-6.

Alternatively, a composition comprising the active agent(s)/activeingredient(s) may be administered by inhalation using a dry powderinhaler (DPI). Such DPIs typically administer the active agent as afree-flowing powder that is dispersed in a subject's air-stream duringinspiration. In order to achieve a free flowing powder, the activeagent(s)/active ingredient(s) is typically formulated with a suitableexcipient such as lactose, starch, mannitol, dextrose, polylactic acid,polylactide-co-glycolide, and combinations thereof. Typically, theactive agent(s)/active ingredient(s) is micronized and combined with anexcipient to form a blend suitable for inhalation. Accordingly, in oneembodiment of the invention, the active agent(s)/active ingredient(s) isin micronized form. For example, a representative composition for use ina DPI comprises dry lactose having a particle size between about 1 μmand about 100 μm (e.g., dry milled lactose) and micronized particles ofthe active agent. Such a dry powder formulation can be made, forexample, by combining lactose with the active agent and then dryblending the components. Alternatively, if desired, the active agent canbe formulated without an excipient. The composition is then typicallyloaded into a DPI, or into inhalation cartridges or capsules for usewith a DPI. DPIs are well known to those of ordinary skill in the art,and many such devices are commercially available, with representativedevices including Aerolizer® (Novartis), Airmax™ (IVAX), ClickHaler®(Innovata Biomed), Diskhaler® (GlaxoSmithKline), Diskus® or Accuhaler(GlaxoSmithKline), Easyhaler® (Orion Pharma), Eclipse™ (Aventis),FlowCaps® (Hovione), Handihaler® (Boehringer Ingelheim), Pulvinal®(Chiesi), Rotahaler® (GlaxoSmithKline), SkyeHaler™ or Certihaler™(SkyePharma), Twisthaler (Schering-Plough), Turbuhaler® (AstraZeneca),Ultrahaler® (Aventis), and the like.

Alternatively, the composition comprising the active agent may beadministered by inhalation using a metered-dose inhaler (MDI). Such MDIstypically discharge a measured amount of the active agent usingcompressed propellant gas. Metered-dose formulations thus typicallycomprise a solution or suspension of the active agent in a liquefiedpropellant, such as a chlorofluorocarbon such as CCl₃F or ahydrofluoroalkane (HFA) such as 1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3-heptafluoro-n-propane (HFA 227), although HFAs aregenerally preferred due to concerns about chlorofluorocarbons affectingthe ozone layer. Additional optional components of HFA formulationsinclude co-solvents, such as ethanol or pentane, and surfactants, suchas sorbitan trioleate, oleic acid, lecithin, and glycerin. See, forexample, U.S. Pat. No. 5,225,183 to Purewal et al., EP 0717987 A2(Minnesota Mining and Manufacturing Company), and WO 92/22286 (MinnesotaMining and Manufacturing Company). A representative composition for usein an MDI comprises from about 0.01-5 wt % of active agent; from about0-20 wt % ethanol; and from about 0-5 wt % surfactant; with theremainder being an HFA propellant. Such compositions are typicallyprepared by adding a chilled or pressurized hydrofluoroalkane to asuitable container containing the active agent, ethanol (if present) andthe surfactant (if present). To prepare a suspension, the active agentis micronized and then combined with the propellant. The formulation isthen loaded into an aerosol canister, which forms a portion of the MDI.MDIs are well known to those of ordinary skill in the art, and many suchdevices are commercially available, with representative devicesincluding AeroBid Inhaler System (Forest Pharmaceuticals), AtroventInhalation Aerosol (Boehringer Ingelheim), Flovent® (GlaxoSmithKline),Maxair Inhaler (3M), Proventil® Inhaler (Schering), Serevent® InhalationAerosol (GlaxoSmithKline), and the like. Alternatively, a suspensionformulation can be prepared by spray drying a coating of surfactant onmicronized particles of the active agent. See, for example, WO 99/53901(Glaxo Group Ltd.) and WO 00/61108 (Glaxo Group Ltd.).

Additional examples of processes of preparing respirable particles, andformulations and devices suitable for inhalation dosing are described inU.S. Pat. No. 5,874,063 to Briggner et al.; U.S. Pat. No. 5,983,956 toTrofast; U.S. Pat. No. 6,221,398 to Jakupovic et al.; U.S. Pat. No.6,268,533 to Gao et al.; U.S. Pat. No. 6,475,524 to Bisrat et al.; andU.S. Pat. No. 6,613,307 to Cooper.

Alternatively, the pharmaceutical compositions may be suitable for oraladministration. Suitable compositions for oral administration may be inthe form of capsules, tablets, pills, lozenges, cachets, dragees,powders, granules; solutions or suspensions in an aqueous or non-aqueousliquid; oil-in-water or water-in-oil liquid emulsions; elixirs orsyrups; and the like; each containing a predetermined amount of theactive agent.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the composition will typicallycomprise the active agent and one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate. Solid dosageforms may also comprise: fillers or extenders, such as starches,microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and/orsilicic acid; binders, such as carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, suchas glycerol; disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and/or sodium carbonate; solution retarding agents, such as paraffin;absorption accelerators, such as quaternary ammonium compounds; wettingagents, such as cetyl alcohol and/or glycerol monostearate; absorbents,such as kaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants may also be presentin the pharmaceutical compositions. Exemplary coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate,carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, and the like. Examples of pharmaceutically acceptableantioxidants include: water-soluble antioxidants, such as ascorbic acid,cysteine hydrochloride, sodium bisulfate, sodium metabisulfate sodiumsulfite and the like; oil-soluble antioxidants, such as ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin,propyl gallate, alpha-tocopherol, and the like; and metal-chelatingagents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol,tartaric acid, phosphoric acid, and the like.

Compositions may also be formulated to provide slow or controlledrelease of the active agent using, by way of example, hydroxypropylmethyl cellulose in varying proportions or other polymer matrices,liposomes and/or microspheres. In addition, the pharmaceuticalcompositions of the invention may contain opacifying agents and may beformulated so that they release the active agent only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes. The activeagent can also be in micro-encapsulated form, if appropriate, with oneor more of the above-described excipients. Suitable liquid dosage formsfor oral administration include, by way of illustration,pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. Liquid dosage forms typically comprisethe active agent and an inert diluent, such as, for example, water orother solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(e.g., cottonseed, groundnut, corn, germ, olive, castor and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Suspensions may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminium metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionsof the invention may be packaged in a unit dosage form. The term “unitdosage form” refers to a physically discrete unit suitable for dosing asubject, i.e., each unit containing a predetermined quantity of theactive agents calculated to produce the desired therapeutic effecteither alone or in combination with one or more additional units. Forexample, such unit dosage forms may be capsules, tablets, pills, and thelike.

Compositions of the invention can also be administered parenterally(e.g., by subcutaneous, intravenous, intramuscular, or intraperitonealinjection). For such administration, the active agents are provided in asterile solution, suspension, or emulsion. Exemplary solvents forpreparing such formulations include water, saline, low molecular weightalcohols such as propylene glycol, polyethylene glycol, oils, gelatin,fatty acid esters such as ethyl oleate, and the like. A typicalparenteral formulation is a sterile pH 4-7 aqueous solution of theactive agents. Parenteral formulations may also contain one or moresolubilizers, stabilizers, preservatives, wetting agents, emulsifiers,and dispersing agents. These formulations may be rendered sterile by useof a sterile injectable medium, a sterilizing agent, filtration,irradiation, or heat.

Compositions of the invention can also be administered transdermallyusing known transdermal delivery systems and excipients. For example,the active agents can be admixed with permeation enhancers, such aspropylene glycol, polyethylene glycol monolaurate, azacycloalkan-2-onesand the like, and incorporated into a patch or similar delivery system.Additional excipients including gelling agents, emulsifiers and buffers,may be used in such transdermal compositions if desired.

By combining RPL554 with a secondary agent, double therapy can beachieved, i.e., PDE3/PDE4 inhibition activity and activity associatedwith the secondary agent (the β₂-adrenergic receptor agonist), in somecases by administering two compositions and in some cases byadministering a single composition containing the active agent and thesecondary agent. In combination therapy, the amount of RPL554 that isadministered, as well as the amount of secondary agents, may be lessthan the amount typically administered in monotherapy.

RPL554 may be either physically mixed with the second active agent (theβ₂-adrenergic receptor agonist) to form a composition containing bothagents; or each agent may be present in separate and distinctcompositions which are administered to the subject simultaneously orsequentially. For example, RPL554 can be combined with a second activeagent using conventional procedures and equipment to form a combinationof active agents comprising RPL554 and a second active agent.Additionally, the active agents may be combined with a pharmaceuticallyacceptable carrier to form a pharmaceutical composition comprisingRPL554, a second active agent and a pharmaceutically acceptable carrier.In this embodiment, the components of the composition are typicallymixed or blended to create a physical mixture. The physical mixture isthen administered in a therapeutically effective amount using any of theroutes described herein.

Alternatively, the active agents may remain separate and distinct beforeadministration to the subject. In this embodiment, the agents are notphysically mixed together before administration but are administeredsimultaneously or at separate times as separate compositions. Suchcompositions can be packaged separately or may be packaged together in akit. When administered at separate times, the secondary agent willtypically be administered less than 24 hours after administration ofRPL554. In other embodiments this timed relationship is less than 12hours, less than 8 hours, less than 6 hours, less than 4 hours, lessthan 3 hours, less than 1 hour, less than thirty minutes, less than tenminutes, less than one minute, or immediately after administration ofRPL554. This is also referred to as sequential administration. Thus,RPL554 can be administered by inhalation simultaneously or sequentiallywith another active agent using an inhalation delivery device thatemploys separate compartments (e.g. blister packs) for each activeagent, where sequential may mean being administered immediately afteradministration of RPL554 or at some predetermined time later (e.g., onehour later or three hours later). Alternatively, the combination may beadministered using separate delivery devices, i.e., one delivery devicefor each agent. Additionally, the agents can be delivered by differentroutes of administration, i.e., one by inhalation and the other by oraladministration.

Typically, the kit comprises a first dosage form comprising RPL554 andat least one additional dosage form comprising one or more of thesecondary agents set forth herein, in quantities sufficient to carry outthe methods of the invention. The first dosage form and the second (orthird, etc,) dosage form together comprise a therapeutically effectiveamount of active agents for the treatment or prevention of a disease ormedical condition in a subject. Secondary agent(s), when included, arepresent in a therapeutically effective amount. i.e., are typicallyadministered in an amount that produces a therapeutically beneficialeffect when co-administered with RPL554. The secondary agent can be inthe form of a pharmaceutically acceptable acid addition salt, solvate,optically pure stereoisomer, and so forth. Thus, secondary agents listedbelow are intended to include all such forms, and are commerciallyavailable or can be prepared using conventional procedures and reagents.Suitable doses for a secondary agent are typically in the range of about0.05 μg/day to about 500 mg/day.

Diseases and Conditions

The combination of (a) a PDE3/PDE4 inhibitor as defined herein and (b) aβ₂-adrenergic receptor agonist as defined herein is useful for treatinga disease or condition which is based on (i) acute or chronicobstruction of vessels or bronchi or (ii) acute or chronic inflammation,in a subject in need thereof.

Typically, the disease or condition is selected from:

1. respiratory tract: obstructive diseases of the airways including:asthma, including bronchial, allergic, intrinsic, extrinsic,exercise-induced, drug-induced (including aspirin and NSAID-induced) anddust-induced asthma, both intermittent and persistent and of allseverities, and other causes of airway hyper-responsiveness; chronicobstructive pulmonary disease (COPD); bronchitis, including infectiousand eosinophilic bronchitis; emphysema; bronchiectasis; cystic fibrosis;sarcoidosis; farmer's lung and related diseases; hypersensitivitypneumonitis; lung fibrosis, including cryptogenic fibrosing alveolitis,idiopathic interstitial pneumonias, fibrosis complicatinganti-neoplastic therapy and chronic infection, including tuberculosisand aspergillosis and other fungal infections; complications of lungtransplantation; vasculitic and thrombotic disorders of the lungvasculature, and pulmonary hypertension; antitussive activity includingtreatment of chronic cough associated with inflammatory and secretoryconditions of the airways, and iatrogenic cough; acute and chronicrhinitis including rhinitis medicamentosa, and vasomotor rhinitis;perennial and seasonal allergic rhinitis including rhinitis nervosa (hayfever); nasal polyposis; acute viral infection including the commoncold, and infection due to respiratory syncytial virus, influenza,coronavirus (including SARS) and adenovirus;2. bone and joints: arthritides associated with or includingosteoarthritis/osteoarthrosis, both primary and secondary to, forexample, congenital hip dysplasia; cervical and lumbar spondylitis, andlow back and neck pain; rheumatoid arthritis and Still's disease;seronegative spondyloarthropathies including ankylosing spondylitis,psoriatic arthritis, reactive arthritis and undifferentiatedspondarthropathy; septic arthritis and other infection-relatedarthopathies and bone disorders such as tuberculosis, including Potts'disease and Poncet's syndrome; acute and chronic crystal-inducedsynovitis including urate gout, calcium pyrophosphate depositiondisease, and calcium apatite related tendon, bursal and synovialinflammation; Behcet's disease; primary and secondary Sjogren'ssyndrome; systemic sclerosis and limited scleroderma; systemic lupuserythematosus, mixed connective tissue disease, and undifferentiatedconnective tissue disease; inflammatory myopathies includingdermatomyositits and polymyositis; polymalgia rheumatica; juvenilearthritis including idiopathic inflammatory arthritides of whateverjoint distribution and associated syndromes, and rheumatic fever and itssystemic complications; vasculitides including giant cell arteritis,Takayasu's arteritis, Churg-Strauss syndrome, polyarteritis nodosa,microscopic polyarteritis, and vasculitides associated with viralinfection, hypersensitivity reactions, cryoglobulins, and paraproteins;low back pain; Familial Mediterranean fever, Muckle-Wells syndrome, andFamilial Hibernian Fever, Kikuchi disease; drug-induced arthalgias,tendonititides, and myopathies;3. pain and connective tissue remodelling of musculoskeletal disordersdue to injury [for example sports injury] or disease: arthitides (forexample rheumatoid arthritis, osteoarthritis, gout or crystalarthropathy), other joint disease (such as intervertebral discdegeneration or temporomandibular joint degeneration), bone remodellingdisease (such as osteoporosis, Paget's disease or osteonecrosis),polychondritits, scleroderma, mixed connective tissue disorder,spondyloarthropathies or periodontal disease (such as periodontitis);4. skin: psoriasis, atopic dermatitis, contact dermatitis or othereczematous dermatoses, and delayed-type hypersensitivity reactions;phyto- and photodermatitis; seborrhoeic dermatitis, dermatitisherpetiformis, lichen planus, lichen sclerosus et atrophica, pyodermagangrenosum, skin sarcoid, discoid lupus erythematosus, pemphigus,pemphigoid, epidermolysis bullosa, urticaria, angioedema, vasculitides,toxic erythemas, cutaneous, eosinophilias, alopecia areata, male-patternbaldness, Sweet's syndrome, Weber-Christian syndrome, erythemamultiforme; cellulitis, both infective and non-infective; panniculitis;cutaneous lymphomas, non-melanoma skin cancer and other dysplasticlesions; drug-induced disorders including fixed drug eruptions;5. eyes: blepharitis; conjunctivitis, including perennial and vernalallergic conjunctivitis; iritis; anterior and posterior uveitis;choroiditis; autoimmune; degenerative or inflammatory disordersaffecting the retina; ophthalmitis including sympathetic ophthalmitis;sarcoidosis; infections including viral, fungal, and bacterial;6. gastrointestinal tract: glossitis, gingivitis, periodontitis;oesophagitis, including reflux; eosinophilic gastro-enteritis,mastocytosis, Crohn's disease, colitis including ulcerative colitis,proctitis, pruritis ani; coeliac disease, irritable bowel syndrome, andfood-related allergies which may have effects remote from the gut (forexample migraine, rhinitis or eczema);7. abdominal: hepatitis, including autoimmune, alcoholic and viral;fibrosis and cirrhosis of the liver; cholecystitis; pancreatitis, bothacute and chronic;8. genitourinary: nephritis including interstitial andglomerulonephritis; nephrotic syndrome; cystitis including acute andchronic (interstitial) cystitis and Hunner's ulcer; acute and chronicurethritis, prostatitis, epididymitis, oophoritis and salpingitis;vulvo-vaginitis; Peyronie's disease; erectile dysfunction (both male andfemale);9. allograft rejection: acute and chronic following, for example,transplantation of kidney, heart, liver, lung, bone marrow, skin orcornea or following blood transfusion; or chronic graft versus hostdisease;10. CNS: Alzheimer's disease and other dementing disorders including CJDand nvCJD; amyloidosis; multiple sclerosis and other demyelinatingsyndromes; cerebral atherosclerosis and vasculitis; temporal arteritis;myasthenia gravis; acute and chronic pain (acute, intermittent orpersistent, whether of central or peripheral origin) including visceralpain, headache, migraine, trigeminal neuralgia, atypical facial pain,joint and bone pain, pain arising from cancer and tumor invasion,neuropathic pain syndromes including diabetic, post-herpetic, andHIV-associated neuropathies; neurosarcoidosis; central and peripheralnervous system complications of malignant, infectious or autoimmuneprocesses;11. other auto-immune and allergic disorders including Hashimoto'sthyroiditis, Graves' disease, Addison's disease, diabetes mellitus,idiopathic thrombocytopaenic purpura, eosinophilic fasciitis, hyper-IgEsyndrome, antiphospholipid syndrome;12. other disorders with an inflammatory or immunological component;including acquired immune deficiency syndrome (AIDS), leprosy, Sezarysyndrome, and paraneoplastic syndromes;13. cardiovascular: atherosclerosis, affecting the coronary andperipheral circulation; pericarditis; myocarditis, inflammatory andauto-immune cardiomyopathies including myocardial sarcoid; ischaemicreperfusion injuries; endocarditis, valvulitis, and aortitis includinginfective (for example syphilitic); vasculitides; disorders of theproximal and peripheral veins including phlebitis and thrombosis,including deep vein thrombosis and complications of varicose veins;14. oncology: treatment of common cancers including prostate, breast,lung, ovarian, pancreatic, bowel and colon, stomach, skin and braintumors and malignancies affecting the bone marrow (including theleukaemias) and lymphoproliferative systems, such as Hodgkin's andnon-Hodgkin's lymphoma; including the prevention and treatment ofmetastatic disease and tumour recurrences, and paraneoplastic syndromes;and,15. gastrointestinal tract: Coeliac disease, proctitis, eosinopilicgastro-enteritis, mastocytosis, Crohn's disease, ulcerative colitis,microscopic colitis, indeterminant colitis, irritable bowel disorder,irritable bowel syndrome, non-inflammatory diarrhea, food-relatedallergies which have effects remote from the gut, e.g., migraine,rhinitis and eczema.

Preferably, the disease or condition is asthma, allergic asthma, hayfever, allergic rhinitis, bronchitis, emphysema, bronchiectasis, chronicobstructive pulmonary disease (COPD), adult respiratory distresssyndrome (ARDS), steroid resistant asthma, severe asthma, paediatricasthma, cystic fibrosis, lung fibrosis, pulmonary fibrosis, interstitiallung disease, skin disorders, atopic dermatitis, psoriasis, ocularinflammation, cerebral ischaemia, or auto-immune diseases.

More preferably, the disease or condition is asthma or chronicobstructive pulmonary disease (COPD).

The subject treated is typically a human.

Typically, the active components (a) and (b) are co-administered.Preferably, the active components (a) and (b) are contained in a singledosage form.

Alternatively, active components (a) and (b) may be administeredseparately. There may be a time delay between the administration of theactive components (a) and (b).

The active components (a) and (b) may be administered by inhalation. Theactive components (a) and (b) may be administered by aerosol.

The amount of active agent administered per dose or the total amountadministered per day may be predetermined or it may be determined on anindividual patient basis by taking into consideration numerous factors,including the nature and severity of the patient's condition, thecondition being treated, the age, weight, and general health of thepatient, the tolerance of the patient to the active agent, the route ofadministration, pharmacological considerations such as the activity,efficacy, pharmacokinetics and toxicology profiles of the active agentand any secondary agents being administered, and the like. Treatment ofa patient suffering from a disease or medical condition (such as COPD)can begin with a predetermined dosage or a dosage determined by thetreating physician, and will continue for a period of time necessary toprevent, ameliorate, suppress, or alleviate the symptoms of the diseaseor medical condition. Patients undergoing such treatment will typicallybe monitored on a routine basis to determine the effectiveness oftherapy. For example, in treating COPD, significant improvement inforced expiratory volume (measured in one second) may be used todetermine the effectiveness of treatment. Similar indicators for theother diseases and conditions described herein, are well-known to thoseskilled in the art, and are readily available to the treating physician.Continuous monitoring by the physician will insure that the optimalamount of active agent will be administered at any given time, as wellas facilitating the determination of the duration of treatment. This isof particular value when secondary agents are also being administered,as their selection, dosage, and duration of therapy may also requireadjustment. In this way, the treatment regimen and dosing schedule canbe adjusted over the course of therapy so that the lowest amount ofactive agent that exhibits the desired effectiveness is administeredand, further, that administration is continued only so long as isnecessary to successfully treat the disease or medical condition.Accordingly, in one embodiment, compositions of the invention are usefulfor treating smooth muscle disorders in mammals, including humans andtheir companion animals (e.g., dogs, cats etc.). Such smooth muscledisorders include, by way of illustration, overactive bladder, chronicobstructive pulmonary disease and irritable bowel syndrome. Typically,suitable doses for treating smooth muscle disorders or other disordersmediated by β₂- and regenic receptors will range from about 0.14μg/kg/day to about 7 mg/kg/day of active agent; including from about0.15 μg/kg/day to about 5 mg/kg/day. For an average 70 kg human, thiswould amount to about 10 μg per day to about 500 mg per day of activeagent.

Typically, compositions of the invention are useful for treatingpulmonary or respiratory disorders, such as COPD or asthma, in mammalsincluding humans, by administering to a patient a therapeuticallyeffective amount of the composition. Generally, the dose for treating apulmonary disorder will range from about 10-1500 μg/day. The term “COPD”is understood by those of ordinary skill in the art to include a varietyof respiratory conditions, including chronic obstructive bronchitis andemphysema, as exemplified by the teachings of Barnes (2000) N. Engl. J.Med. 343:269-78, and references cited therein.

When administered by inhalation, compositions of the invention typicallyhave the effect of producing bronchodilation. Accordingly, in another ofits method aspects, the invention is directed to a method of producingbronchodilation in a patient, comprising administering to a patient abronchodilation-producing amount of a composition of the invention.Generally, the therapeutically effective dose for producingbronchodilation will range from about 10-1500 μg/day.

Alternatively, compositions of the invention may be used to treatoveractive bladder. When used to treat overactive bladder, a typicaldose will range from about 1.0-500 mg/day. Alternatively, compositionsof the invention may be used to treat irritable bowel syndrome. Whenused to treat irritable bowel syndrome, compositions of the inventionwill typically be administered orally or rectally, and a typical dosewill range from about 1.0-500 mg/day.

It is a finding of the invention, following safety studies, that RPL554does not interact adversely with β₂-adrenergic receptor agonists (suchas salbutamol) with respect to blood pressure or heart rate. Likewise,the cardiovascular effects of β₂-adrenergic receptor agonists (such assalbutamol) are not affected by RPL554.

The following Examples illustrate the invention.

EXAMPLES Example 1 Material and Methods

Preparation of Tissues

Regions of macroscopically normal lungs were taken from uninvolved areasresected from 24 subjects (11 male and 13 female, 60.1±1.6 years old)undergoing lobectomy surgery for lung cancer, but without a history ofchronic airway disease.

Airways were immediately placed into oxygenated Krebs-Henseleit buffersolution (KH) (mM: NaCl 119.0, KCl 5.4, CaCl₂ 2.5, KH₂PO₄1.2, MgSO₄ 1.2,NaHCO₃ 25.0, glucose 11.7; pH 7.4) containing the cyclooxygenase (COX)inhibitor indomethacin (5.0 μM), and transported at 4° C. from the“Regina Elena National Cancer Institute” or the “Sant'Andrea Hospital”to the Respiratory Research Laboratory in the Surgery and MedicineFaculty of “Tor Vergata University”, Rome, Italy. None of the subjectswere chronically treated with theophylline, β₂-agonists orglucocorticosteroids. Serum IgE levels determined on the day of surgerywere in the normal range. Preoperative lung function parameters weregenerally normal and there were no signs of respiratory infections.

In the laboratory, airways were dissected from connective and alveolartissues. Then, segmental bronchi were isolated and stored overnight inKH buffer solution at refrigeration temperature. The next morning,bronchi were cut into rings (n=120; thickness: 1-2 mm; diameter: 5-7 mm)and transferred into 4400 four-chamber 10 ml Isolated Organ Baths (UgoBasile, VA—Italy) containing KH buffer (37° C.) and continuously aeratedwith a 95:5% mixture of O₂/CO₂.

Preparation of Drugs

The following drugs were used: acetylcholine, histamine, salbutamol,papaverine and indomethacin. All substances were obtained fromSigma-Aldrich (St. Louis, USA). Drugs were dissolved in distilled waterexcept for indomethacin and quinine, which were dissolved in ethanol andthen diluted in a KH buffer. The maximal amount of ethanol (0.02%) didnot influence isolated tissue responses (Freas et al., 1989; Hatake andWakabayashi, 2000). RPL554 was kindly provided by Verona Pharma PLC,London, UK. Compounds were stored in small aliquots at −80° C. untiltheir use.

Tension Measurement

Human bronchi were placed in organ baths containing KH buffer solution(37° C.) medicated with indomethacin (5.0 μM), bubbled with 95% O₂/5%CO₂ and suspended under passive tension (0.5-1.0 g). Bronchial ringswere mounted on hooks in the organ baths where one hook was attachedwith threaded to a stationary rod and the other hook tied with thread toan isometric force displacement transducer. Airways were allowed toequilibrate for 90 min with repeated changes of the medicated KH buffersolution every 10 min. Changes in isometric tension were measured with atransducer (Fort 10 WPI, Basile, Instruments, Italy) and the tissueresponsiveness was assessed by acetylcholine (100 μM); when the responsereached a plateau, rings were washed three times and allowed toequilibrate for 45 min.

Study Design

Influence of RPL554 on Electrical Field Stimulation

Each organ bath was fitted with two platinum plate electrodes (1 cm²)placed alongside the tissue (10 mm apart) for electrical fieldstimulation (EFS). Experiments were performed using trains of 10 Hz EFS(biphasic pulse with a constant current of 10V, 0.5 ms, 10 s), one pulseevery 5 min for the first hour and then at 30 min intervals for the next5 hours by a 3165 multiplexing pulse booster (Ugo Basile, VA—Italy)(Binks et al., 2001). After the start of the EFS trains, tissues wereincubated with RPL554 (10 or 100 μM) until maximum inhibition of thecontractile response to electrical field stimulation (EFS) was achieved.Incubation with drug was then terminated and the tissues repeatedlywashed over a 30 min period and then once every 30 min up to 5 h postdrug administration.

Relaxant Effect of RPL554 on Passively Sensitized Bronchi

Human isolated bronchial rings were rotated overnight at roomtemperature in tubes containing KH buffer solution in the absence(non-sensitized control rings) or the presence of 10% vol⁻¹ sensitizingserum (sensitized rings) as described elsewhere (Watson et al., 1997;Rabe, 1998). Subjects suffering from atopic asthma (total IgE>250 U⁻¹specific against common aeroallergens) during exacerbation providedsigned consent for serum donation.

Sera was prepared by centrifugation of whole blood and sera samples werefrozen at −80° C. in 200 ml aliquots until required.

The next morning, after removal of adhering alveolar and connectivetissues, bronchial rings were transferred into an organ bath containingKH buffer (37° C.) and continuously gassed with a 95% O2/5% CO2. Tissueswere pre-incubated for 30 min with RPL554 (1, 10 and 100 μM) and thenfollowed (without washing) by the construction of concentrationresponses curve to histamine (10 nM-1 mM) in the presence of RPL554.

Synergistic Effect of RPL554 Plus β₂-Adrenergic Receptor Agonist

To test the possible synergistic relaxation induced by RPL554 incombination with a β₂-adrenergic receptor agonist (salbutamol), thebronchial rings were contracted with acetylcholine at the concentrationrequired to cause a 70% maximal effect (EC70) and allowed a 15 minstabilization period. Then, concentration response curves wereconstructed to test individual compound RPL554 and β₂-adrenergicreceptor agonist alone; as well as RPL554 administered in combinationwith a β₂-adrenergic receptor agonist in order to produce isobolargraphs as described elsewhere (Greco et al., 1995; Tallarida, 2001;Goldoni and Johansson, 2007; Boik et al., 2008; Lee, 2010).

Intervals of 20 min between successive concentrations were used to reacha stable level of relaxation before the administration of the nextconcentration. At the completion of the experiment, papaverine (500 μM)was added to relax the tissues completely and provide a standard towhich the relaxation of each tissue could be compared.

Analysis of Results

Analysis of EFS Studies

Bronchial contractile tension induced by EFS was measured as apercentage of control bronchi, and polynomial curves were constructed byfitting models of biological data using nonlinear regression asdescribed elsewhere (Motulsky and Christopoulos, 2004). The maximaleffect (Emax) was identified as the lowest contractile force induced byEFS stimulation and the offset (t_(1/2), min) indicates the time toevoke a half of maximal relaxation. For every three bronchial ringsmounted in the isolated organ bath system, one was used as a timecontrol as described elsewhere (Mercier et al., 2002).

Analysis of Concentration Response Studies

Appropriate curve-fitting to a sigmoidal model was used to calculate theeffect (E), the Emax and the concentration required to cause a 50%maximal effect (EC50). The equation used was log [agonist] vs. response,Variable slope, expressed as Y=Bottom+(Top−Bottom)/{1+10^[(LogEC50−X)*HillSlope]} (Motulsky and Christopoulos, 2004; Goodman et al.,2008). E/Emax was expressed as percentage of Emax elicited by thecontractile agents; EC50 values were converted to pD₂ for statisticalanalysis (Goodman et al., 2008) and the relaxant responses wereexpressed as a percentage of papaverine (500 μM) induced relaxation.

Analysis of Synergism Studies

The analysis of the potential synergism between RPL554 plus aβ₂-adrenergic receptor agonist was measured by applying the Berenbaummethod, the Bliss Independence (BI) criterion and the Loewe Additivity(LA) model through curved isoboles (Berenbaum, 1977; Greco et al., 1995;Grabovsky and Tallarida, 2004; Tallarida, 2006; Goldoni and Johansson,2007; Tallarida and Raffa, 2010).

In order to apply the Berenbaum method, the Interaction Index for theEC50 values was evaluated and, therefore, if the Interaction Index was<1 the effect was considered synergistic; if the Interaction Indexwas >1 the effect was antagonistic and if the Interaction Index was=0the effect was considered additive (Goldoni and Johansson, 2007; Lee,2010).

The BI theory for two agents is expressed by the following equation:E(x,y)=Ex+Ey−(Ex*Ey), where E is the fractional effect, and x and y arethe concentrations of two compounds in a combination experiment. If thecombination effect is higher than the expected value from the aboveequation, the interaction is considered synergistic, while if thiseffect is lower, the interaction is antagonistic. Otherwise, the effectis additive and there is no interaction (Greco et al., 1995; Meletiadiset al., 2003; Boucher and Tam, 2006; Goldoni and Johansson, 2007; Boiket al., 2008; Lee, 2010). In this study, the BI equation wascharacterized by X=RPL554 and Y=β₂-adrenergic receptor agonist.

Control concentration response curves for salbutamol and RPL554 frombronchi from each lung were fitted to a 4 parameter logistic equation tocalculate parameter estimates of Emax, slope (nH) and potency (EC50).The following parameter estimates Emax and nH (mean±SD) and EC50(geomean, 95% CI) for salbutamol (78±11, 1.572±0.482, 0.283(0.064-1.239) μM, n=5, respectively) and RPL554 (100±0, 2.271±0.713,21.2 (11.5-39.1)μM, n=5, respectively) were then used to calculate theadditive response for the drug pair combination to evaluate synergism(Tallarida and Raffa 2010)(Grabovsky and Tallarida 2004). Using theconcept of dose equivalence, the relationship a/A+b/B=1 was reformulatedas b+beq (a)=B, where beq is the dose equivalent of a and solving forbeq(a) by equating the two individual concentration response curvesE_(A)=f(A) and E_(B)=f(B). The additive response (E_(ab)) for each dosecombination with respect to B was then calculated by insertion of B intoE_(B)=f(B). The difference between the observed relaxation response tothe combination doses and the additive response was calculated andanalysed using a one sample t-test and for multiple comparisons, theprobability was adjusted for multiple comparisons using a Bonferronicorrection. For illustrative purposes, the 1:1 dose combinations wereanalysed for synergy.

Statistical Analysis

All values are presented as mean±SEM for each treatment group.Statistical significance was assessed by Student's t test or analysis ofvariance (ANOVA) if required and the level of statistical significancewas defined as P<0.05 (Motulsky, 1995). All data analyses were performedusing computer software (GraphPad Prism, San Diego Calif. USA; MicrosoftExcel, Redmond Wash. USA).

Synergistic Effect of RPL554 Administered in Combination with Salbutamol(a β₂-Adrenergic Receptor Agonist)

To test the synergistic relaxation induced by RPL554 administered incombination with salbutamol, the bronchial rings were contracted withacetylcholine at the concentration required to cause a 70% maximaleffect (EC70) and allowed a 15 min stabilization period. Then,concentration response curves were constructed to test individualcompound RPL554, or salbutamol alone; or RPL554 administered incombination with salbutamol in order to produce isobolar graphs(salbutamol:RPL554 and ranging from 10:1 to 1:100) as describedelsewhere (Greco et al., 1995; Tallarida, 2001; Goldoni and Johansson,2007; Boik et al., 2008; Lee, 2010).

Intervals of 20 min between successive concentrations were used to reacha stable level of relaxation before the administration of the nextconcentration. At the completion of the experiment, papaverine (500 μM)was added to relax the tissues completely and provide a standard towhich the relaxation of each tissue could be compared.

Results

Baseline Characteristics of Bronchial Rings

There were no significant differences (P>0.05) between the baselinecharacteristics of the human isolated bronchial rings employed in thestudy concerning the wet weight (220.5±16.5 mg), the contraction inducedby acetylcholine (100 μM) (440±95 mg) and the contraction induced by EFS(10 Hz) before treatments with drugs (445±98 mg).

In preliminary experiments, a concentration response curve toacetylcholine (from 1 nM to 1 mM) was constructed to establish asub-maximal response (approximately 70% maximum response; 1250±190 mg;n=5) for subsequent studies.

Influence of RPL554 on Bronchial Tone of Isolated Human Airways

RPL554 inhibited the contractile response induced by EFS of humanbronchial tissues that was maintained for at least 5 h after exposure tothis drug (FIG. 1). RPL554 abolished these contractile responses at amaximum concentration of 100 μM (Emax 91.33±3.37%; T_(1/2) 23 0.7±12.3min).

RPL554 caused a concentration-dependent relaxation of human isolatedbronchial tissues pre-contracted with acetylcholine. RPL554 was lesspotent (P<0.05) than salbutamol in bronchial relaxation but, in contrastto salbutamol, RPL554 completely relaxed tissues (P<0.001). Controlconcentration response curves for salbutamol and RPL554 from bronchifrom each lung were fitted to a 4 parameter logistic equation tocalculate parameter estimates of Emax, slope (nH) and potency (EC50).The following parameter estimates Emax and nH (mean±SD) and EC50(geomean, 95% CI) for salbutamol (78±11, 1.572±0.482, 0.283(0.064-1.239)μM, n=5, respectively) and RPL554 (100±0, 2.271±0.713, 21.2(11.5-39.1)μM, n=5, respectively) (FIG. 2).

The passive sensitization of bronchi enhanced the contractile effect ofhistamine compared to non-sensitized tissues. In passively sensitizedbronchi, RPL554 at 1 and 10 μM significantly (P<0.001) shifted leftwardthe concentration response curve to histamine compared with untreatedtissues and RPL554 at 100 μM completely abolished the contractioninduced by histamine (FIG. 3, Table 1).

TABLE 1 Effect of RPL554 on contraction induced by histamine inpassively sensitized bronchi. Data shown are from experiments performedwith samples of n = 5 different subjects and they are represented asmean ± SEM ***P < 0.001 vs passively sensitized control. Passivelysensitized Non- RPL554 RPL554 sensitized Control RPL554 1 uM 10 uM 100uM Emax 100.7 ± 1.7 101.8 ± 1.4  70.3 ± 2.7***  58.1 ± 2.2***  nd pD2 4.82 ± 0.03*** 5.29 ± 0.03 4.97 ± 0.07*** 4.98 ± 0.07*** ndSynergistic Relaxant Effect of RPL554 Plus Salbutamol on Human BronchialTone

The BI study indicated that the interaction of RPL554 plus salbutamolinduced a significant synergistic relaxant effect for RPL554 at 1 and 10μM (both P<0.05) on the human bronchial tone pre-contracted withacetylcholine and the maximal synergism was detected for RPL554 at 1 μMplus salbutamol at 100 nM (BI delta effect: 0.29±0.11), although therewas a less significant synergistic interaction between salbutamol andRPL554 at lower concentrations (10 nM and 100 nM, P>0.05) (FIG. 4). Infact, the BI analysis of the isomolar association (1:1) of RPL554 plussalbutamol only showed a signal for synergism (P=0.08) based on theenhancement of the relaxant potency (Table 2). Nevertheless, theBerenbaum analysis demonstrated that RPL554 plus salbutamol elicited asynergistic interaction for RPL554 over the concentration range of 10 nMto 10 μM (Interaction Index: 0.25±0.06) and that RPLL554 significantlycaused a leftward shift of the relaxant concentration response curves tosalbutamol of 0.89±0.14 logarithms (P<0.05).

TABLE 2 Relaxant synergistic effect of RPL554 plus salbutamol (isomolar,1:1) on sub-maximal contraction induced by acetylcholine. Data shown arefrom experiments performed with samples of n = 5 different subjects andthey are represented as mean ± SEM. ** P < 0.01 for zero-interactionhypothesis delta effect (observed vs expected values). RPL554 +salbutamol Observed Expected Emax 96.31 ± 3.43  94.66 ± 4.01 pD2 6.78 ±0.30  6.33 ± 0.15 Delta potency 0.45 ± 0.02 (observed-expected)

Finally, the 3D surface analysis using the BI method demonstrated thatsalbutamol induced a synergistic interaction extended acrossconcentrations when administered in association with RPL554 (FIG. 5).The observed and additive relaxation response for the 1:1 dosecombinations of salbutamol and RPL554 synergy are shown in FIG. 6.

It has been demonstrated that the selective inhibition of PDE3/PDE4 byRPL554 elicited relaxation of bronchial tone in human isolated airwayswhich extends and supports observations previously reported inguinea-pig isolated trachea (Boswell-Smith et al., 2006b). Thisinhibitory effect was maintained for up to 5 h after termination of drugexposure, confirming the long duration of action of this compound inhuman airways. Furthermore, RPL554 acted to relax airways contractedwith either histamine or acetylcholine. Moreover, prior incubation oftissues with RPL554 resulted in a significant protection of the tissuesagainst the contractile action of exogenously administered histamine inpassively sensitized bronchi. In addition, the inhibition of PDE3/4associated with a β₂-adrenergic receptor agonist (salbutamol)demonstrated a synergistic effect on relaxation of ASM. These resultsshow that RPL554 is a good functional antagonist against contractileagents in human bronchial tissues and when combined with a β₂-adrenergicreceptor agonist can have the ability to provide further synergisticbronchodilation.

RPL554 caused a concentration and time dependent inhibition ofcontractile responses elicited by EFS which had a considerably longerduration of action against EFS-induced contractile responses than otherPDE4 inhibitors (Spina et al., 1998; Boswell-Smith et al., 2006b).

RPL554 was particularly effective at inhibiting the contractile responsein passively sensitized human bronchi contracted with histamine and avariety of selective PDE3 and PDE4 inhibitors have been reported tosignificantly attenuate acute bronchospasm induced by antigen insensitized guinea pigs (Boswell-Smith et al., 2006a).

RPL554 also induced a noticeable decrease in the maximum response tohistamine in passively sensitized bronchi.

Safety Study of the Combination of the Present Invention

This study was undertaken to determine whether RPL554 has cardiovascularinteractions with a β₂-adrenergic receptor agonist (salbutamol). Amuscarinic receptor antagonist (atropine) was included for completeness.RPL554 is a dual PDE3/PDE4 inhibitor being developed for treatment ofchronic obstructive pulmonary disease (COPD) and asthma as an inhaledbronchodilator with possible anti-inflammatory actions. Cardiovascularresponses to RPL554, salbutamol and atropine, given as intravenous bolusinjection, were assessed as cardiovascular changes, measured as peakpost-injection changes in blood pressure and heart rate. The studydesign was blind and random with drugs given as pairs five minutes apartin an alternating manner, e.g., RPL554 followed by salbutamol and viceversa. The doses chosen for study produced cardiovascular effects, andpresumably plasma concentrations, much higher than those used forinhalation. Instead they were chosen to test for possible interactionsunder supra therapeutic conditions.

Summary

In this study, the effects of intravenously administered RPL554,salbutamol and atropine, alone and in combinations, were examinedaccording to a randomized design, on blood pressure and heart rate inanaesthetized rats. Doses producing a 15-30 mmHg increase in mean bloodpressure (MBP) and 30-60 beats per minute increase in heart rate (HR)were chosen from an initial dose-response study for RPL554 andsalbutamol. For atropine, which produced lesser effects on bloodpressure or heart rate, the maximum dose administered was chosen forfurther study. Thereafter, the chosen doses were examined in pairsadministered 5 minutes part. The data from the study suggest that therewas no interaction in cardiovascular terms between RPL554 andsalbutamol, or between RPL554 and atropine in terms of effects on MBP orHR in anaesthetized rats. In order to rigorously test for possibleinteractions, the doses and route of administration chosen for studyproduced cardiovascular effects not seen with the usual therapeuticdoses. In addition, the bolus intravenous route presumably resulted inmuch higher plasma concentrations than those not seen with the lowertherapeutic doses given by inhalation. The intention of the study was touse supra normal conditions in order to challenge for possibleinteractions under supra therapeutic conditions.

Protocol

The studies were performed on Sprague-Dawley male rats from CharlesRiver weighing 200-250 grms. Once the rats had been delivered to theexperimental laboratory they did not have access to food and water for1-3 hours. For the purposes of the experiment the animals wereanaesthetized with thiobutabarbital at a dose of 100 mg/kg given by thei.p. route. There was no necessity for supplemental anaesthesia and atthe end of the experimental period each rat was sacrificed. The studyhad two parts: (I) an initial dose ranging study and (II) theinteraction study. The drugs studied were: RPL554 (R), salbutamol (S),atropine (A) all dissolved in saline.

(I) Initial Dose-Ranging Study

An initial dose-ranging study was performed as follows: cumulativedose-response curves for intravenous bolus doses of the three drugs wereperformed to determine appropriate doses of each of the three drugs forthe full study. Doses of RPL554 or salbutamol producing a 15-30 mmHgincrease in mean blood pressure (MBP) and 30-60 beats per minuteincrease in heart rate (HR) were chosen from this initial dose-responsestudy. Atropine produced limited cardiovascular effects and so a highdose was chosen as being one for which there is literature evidence ofprofound muscarinic receptor blockade. This dose-ranging study involvedfour rats in which cumulative doses of each of the 3 drugs were given ona dose-doubling basis. Doses of each of the drugs were given as i.v.bolus injections every 5 minutes with 1 hour between different drugs.The order of injections were: for animal 1—S, R, A; animal 2—R, A, S;animal 3—A, S, R and animal 4—R, A, S. As indicated above the doses androutes of injection where chosen as supernormal and above dosesproducing effects when given by inhalation.

From this study the following doses chosen for the subsequentinteraction study:

R=8 μg/kg—8 μg/mL in 0.9% saline

S=2 μg/kg—2 μg/mL in 0.9% saline

A=32 μg/kg—32 μg/mL in 0.9% saline

(II) Interaction Study

In this study all drugs were given as a single dose by i.v. bolusinjection, via a jugular vein cannula, and at a volume of 1 mL/kg. Thestudy design was blind and random with three lines each containing fouranimals for a total of 12 animals.

Pairs of drugs were given to individual animals on a randomized blindbasis using a line by line design where each line contained four ratswith injections as follows: R1S2, S1R2, R1A2 and A1R2 (in random orderwhere 1 indicates the first drug given and 2 indicates the second druggiven 5 minutes after the first drug). A total of 3 lines were studiedthus a total of 12 rats were used and none of the animals had to bereplaced. The study was stopped after 12 animals for the purposes of aprovisional analysis to determine if further study was warranted and, ifso, were more lines and/or dose adjustments required (an adaptivedesign).

The blood pressure was measured from a carotid artery using a transducerwhose output was processed by AD Instruments PowerLab 26T. The heartrate was calculated on a beat to beat basis from the ECG and bloodpressure traces. Analysis was by LabChart 6. Measurements were madeusing the functions in LabChart 6.

All drug effects were measured at the time of peak response to eachinjection of drug. The time to peak response with salbutamol wasapproximately 20 seconds for blood pressure and 60 seconds for heartrate. For RPL554 the corresponding times were 60-80 seconds for bloodpressure and 2-4.5 minutes for heart rate. For atropine, it was 4minutes for both blood pressure and heart rate.

Data were recorded as systolic, diastolic and calculated mean bloodpressure in mmHg while heart rate was recorded as beats/min. The valuesrecorded were processed to provide drug-induced changes in bloodpressure and heart rate from two control values. One of these was priorto the first injection and the second prior to the second injection. Thecalculated changes were also normalized to these two control values andexpressed as either a negative value for a fall from control values oras positive for an increase from control values.

Results

Primary Pharmacodynamics

TABLE 3 Effects of RPL554, salbutamol and atropine, alone and afteranother drug, on changes from control (Δ) mean arterial blood pressureand mean heart rate in anaesthetized rats (n = 3 for each mean value)ΔMBP ΔHR (mmHg and %) (bpm and %) Effect of RPL554 when paired withsalbutamol or atropine RPL554 before salbutamol* −15 −15% 36 10% RPL554after salbutamol** −17 −16% 29 7% RPL554 before atropine* −17 −16% 30 8%RPL554 after atropine** −19 −20% 33 10% Effect of salbutamol when pairedwith RLP554 Salbutamol before −32 −30% 58 18% RPL554* Salbutamol after−23 −24% 43 11% RPL554*** Effect of atropine when paired with RPL554Atropine before RPL554* −5.0 −5% 6.3 1% Atropine after RPL554**** −2.7−3% 21 5%

Drugs were given as i.v. bolus injections in pairs 5 minutes apart. Avalues are the difference in peak effects from the pre-drug value,expressed as change or percentage change with respect to pre-drugvalues. *=control response to drug with no prior drug treatment

The primary pharmacodynamic variables measured were systolic anddiastolic blood pressure with computed mean blood pressure, and heartrate. The summary of the changes seen are shown in Table 3 above.

As can be seen in all cases, at the dose studied, RPL554 produced a fallin blood pressure, regardless of whether systolic, diastolic or meanpressures were expressed as actual changes, or normalized for pre-drugvalues. When measured before or after the prior administration ofsalbutamol or atropine, changes in blood pressure to RPL554 injectionwere not different, as can be seen in Table 3, whether changes wereexpressed as changes from pre-drug, or were normalized as a percentage.

Heart rate responses were similarly not influenced by the prioradministration of the other drugs although the administration of thesecond drug 5 minutes after the first did change pre-drug values in amanner that depended on the drug considered.

Secondary Pharmacodynamics and Safety Pharmacology

The secondary pharmacodynamic variables were: ECG, respiratory rate andarrhythmias. No significant changes were seen in the ECG or respiratoryrate, and no arrhythmias were seen.

Pharmacodynamic Drug Interactions

Possible cardiovascular interactions between the three drugs were theprimary aim of the study. No major interactions between the drugs wereseen as presented in Table 3 or seen by inspection of the actualexperimental records.

Example 2

In Vivo Synergistic Effect of RPL554 Administered in Combination withSalbutamol (a β₂-Adrenergic Receptor Agonist)

This Example investigates the ability of RPL554 to reverse thebronchoconstriction induced by bombesin and potential synergisticeffects when RPL554 is administered in combination with salbutamol.

Guinea pigs were anaesthetized and ventilated. Airway obstruction wasinduced by the intravenous administration of bombesin (2 μg/ml; 5ml/hr). Bronchodilation was induced by the iv. administration of RPL554alone at various doses, or in combination with sub maximal dose ofsalbutamol (20 μg/kg). Doses were selected following studies generatinga dose-response curve for both salbutamol to select a dose resulting inapproximately 20% reduction in airways obstruction. Total lungresistance (R_(L)) and mean arterial blood pressure were measured. Dataare expressed as % reduction in airways obstruction or blood pressure.

RPL554 caused a dose-dependent relaxation of guinea pig airways from10-80 μg/kg. In combination with 20 μg/kg salbutamol (a dose that caused34.2±11.1% reduction in airways obstruction), RPL554 also resulted ingreater relaxation of the airways (Table 4). The iv. administration(which was not potential when co-administered with either) of 20 μg/kgRPL554 caused a reduction in mean arterial blood pressure (control:37.3±6.7%; +20 μg/kg salbutamol: 23.3±13.0%).

TABLE 4 % Reduction in airways obstruction RPL554 control RPL554 +salbutamol (20 μg/kg) 10 μg/kg  7.4 ± 1.9 63.7 ± 22.4 20 μg/kg 24.6 ±4.3 66.0 ± 12.9 40 μg/kg 55.2 ± 6.2 76.3 ± 17.4 80 μg/kg 65.1 ± 5.3 77.6± 6.3 

The results provide further evidence that RPL554 is an effectivebronchodilator which, when combined with the β₂-adrenergic receptoragonist salbutamol, has synergistic activities as a bronchodilator, butdoes not interact with the drug classes on blood pressure.

Example 3 Synergistic Effect of RPL554 Administered in Combination withSalmeterol (a β₂-Adrenergic Receptor Agonist)

The method of Example 1 for evaluating the synergistic effect of thecombination of RPL554 and salbutamol is repeated for the β₂-adrenergicreceptor agonist salmeterol. Concentration response curves areconstructed to test RPL554 alone, salmeterol alone, and RPL554administered in combination with salmeterol. The Berenbaum Method andthe Bliss Independence criteria are used to evaluate synergistic actionbetween salmeterol and RPL554. A synergistic effect is observed for thecombination of RPL554 and salmeterol.

Example 4 Synergistic Effect of RPL554 Administered in Combination withFormoterol (a β₂-Adrenergic Receptor Agonist)

The method of Example 1 for evaluating the synergistic effect of thecombination of RPL554 and salbutamol is repeated for the β₂-adrenergicreceptor agonist formoterol. Concentration response curves areconstructed to test RPL554 alone, formoterol alone, and RPL554administered in combination with formoterol. The Berenbaum Method andthe Bliss Independence criteria are used to evaluate synergistic actionbetween formoterol and RPL554. A synergistic effect is observed for thecombination of RPL554 and formoterol.

Example 5 Synergistic Effect of RPL554 Administered in Combination withPirbuterol (a β₂-Adrenergic Receptor Agonist)

The method of Example 1 for evaluating the synergistic effect of thecombination of RPL554 and salbutamol is repeated for the β₂-adrenergicreceptor agonist pirbuterol. Concentration response curves areconstructed to test RPL554 alone, pirbuterol alone, and RPL554administered in combination with pirbuterol. The Berenbaum Method andthe Bliss Independence criteria are used to evaluate synergistic actionbetween pirbuterol and RPL554. A synergistic effect is observed for thecombination of RPL554 and pirbuterol.

Formulation Example 1 Administration by a DPI

RPL554 combination (0.2 mg) is micronized and then blended with lactose(25 mg). This blended mixture is then loaded into a gelatin inhalationcartridge. The contents of the cartridge are administered using a DPI,for example.

A micronized RPL554 combination (100 mg) is blended with milled lactose(25 g) (e.g., lactose in which not greater than about 85% of theparticles have a MMD of about 60 μm to about 90 μm and not less than 15%of the particles have a MMD of less then 15 μm). The blended mixture isthen loaded into individual blisters of a peelable blister pack in anamount sufficient to provide about 10-500 μg of the RPL554 combinationper dose. The contents of the blisters are administered using a DPI.

Alternatively, a micronized RPL554 combination (1 g) is blended withmilled lactose (200 g) to form a bulk composition having a weight ratioof compound to milled lactose of 1:200. The blended composition ispacked into a DPI capable of delivering between about 10-500 μg of theRPL554 per dose.

Alternatively, a micronized RPL554 (100 mg) and a micronizedβ₂-adrenergic receptor agonist (500 mg) are blended with milled lactose(30 g). The blended mixture is then loaded into individual blisters of apeelable blister pack in an amount sufficient to provide about 10 μg toabout 500 μg of the RPL554 per dose. The contents of the blisters areadministered using a DPI.

Formulation Example 2 Compositions for Use in an MDI

A micronized RPL554 combination (10 g) is dispersed in a solutionprepared by dissolving lecithin (0.2 g) in demineralized water (200 mL).The resulting suspension is spray dried and then micronized to form amicronized composition comprising particles having a mean diameter lessthan about 1.5 The micronized composition is then loaded into MDIcartridges containing pressurized 1,1,1,2-tetrafluoroethane in an amountsufficient to provide about 10 μs to about 500 μg of the RPL554 per dosewhen administered by the MDI. Alternately, a suspension containing 5 wt% RPL554 combination, 0.5 wt % lecithin, and 0.5 wt % trehalose isprepared by dispersing 5 g of a RPL554 combination as micronizedparticles with mean size less than 10 μm in a colloidal solution formedfrom 0.5 g of trehalose and 0.5 g of lecithin dissolved in 100 mL ofdemineralized water. The suspension is spray dried and the resultingmaterial is micronized to particles having a mean diameter less than 1.5μm. The particles are loaded into canisters with pressurized1,1,1,2-tetrafluoroethane.

Formulation Example 3 Composition for Use in a Nebulizer Inhaler

RPL554 combination (25 mg) is dissolved in citrate buffered (pH 5)isotonic saline (125 mL). The mixture is stirred and sonicated until thecompound is dissolved. The pH of the solution is checked and adjusted,if necessary, to pH 5 by slowly adding aqueous 1N sodium hydroxide. Thesolution is administered using a nebulizer device that provides about 10μg to about 500 m of the RPL554 per dose.

Formulation Example 4 Hard Gelatin Capsules for Oral Administration

RPL554 combination (50 g), spray-dried lactose (440 g) and magnesiumstearate (10 g) are thoroughly blended. The resulting composition isthen loaded into hard gelatin capsules (500 mg of composition percapsule).

Formulation Example 5 Suspension for Oral Administration

The following ingredients are mixed to form a suspension containing 100mg of compound per 10 mL of suspension:

Ingredients Amount RPL554 combination 1.0 g Fumaric acid 0.5 g Sodiumchloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 g Granulatedsugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum^(RTM) K (magnesiumaluminum silicate) 1.0 g Flavoring 0.035 mL Colorings 0.5 mg Distilledwater q.s. to 100 mL

Formulation Example 5 Injectable Formulation for Administration byInjection

RPL554 combination (0.2 g) is blended with 0.4 M sodium acetate buffersolution (2.0 mL). The pH of the resulting solution is adjusted to pH 4using 0.5 N aqueous hydrochloric acid or 0.5 N aqueous sodium hydroxide,as necessary, and then sufficient water for injection is added toprovide a total volume of 20 mL. The mixture is then filtered through asterile filter (0.22 micron) to provide a sterile solution suitable foradministration by injection.

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The invention claimed is:
 1. A nebulizer inhaler, a dry powder inhaleror a metered dose inhaler comprising: a composition comprising (a) aPDE3/PDE4 inhibitor which is9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-oneor a pharmaceutically acceptable acid addition salt thereof and (b) aβ₂-adrenergic receptor agonist; and one or more pharmaceuticallyacceptable carriers, diluents, or excipients.
 2. A nebulizer inhaler, adry powder inhaler or a metered dose inhaler according to claim 1, inwhich the PDE3/PDE4 inhibitor (a) is9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one.3. A nebulizer inhaler, a dry powder inhaler or a metered dose inhaleraccording to claim 1, in which the β₂-adrenergic receptor agonist (b) issalbutamol, bitolterol, fenoterol, formoterol, indacaterol, isoetharine,levalbuterol, metaproterenol, pirbuterol, salmefamol, salmeterol orterbutaline.
 4. A nebulizer inhaler, a dry powder inhaler or a metereddose inhaler according to claim 1, in which β₂-adrenergic receptoragonist (b) is salbutamol, salmeterol, formoterol, or pirbuterol.
 5. Anebulizer inhaler, a dry powder inhaler or a metered dose inhaleraccording to claim 1, wherein the composition is a fixed combination. 6.A method of treating a disease or condition, in a subject in needthereof, which method comprises administering to said subject (a) aPDE3/PDE4 inhibitor which is9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-oneor a pharmaceutically acceptable acid addition salt thereof, and (b) aβ₂-adrenergic receptor agonist, wherein the disease or condition isselected from the group consisting of asthma, allergic asthma, hayfever, allergic rhinitis, bronchitis, emphysema, bronchiectasis, chronicobstructive pulmonary disease (COPD), adult respiratory distresssyndrome (ARDS), steroid resistant asthma, severe asthma and paediatricasthma.
 7. A method according to claim 6, which method comprisesadministering to said subject a therapeutically effective, non-toxicamount of a composition comprising (a) a PDE3/PDE4 inhibitor which is9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-oneor a pharmaceutically acceptable acid addition salt thereof and (b) aβ₂-adrenergic receptor agonist.
 8. A method according to claim 6,wherein the disease or condition is asthma or chronic obstructivepulmonary disease (COPD).
 9. A method according claim 6, wherein saidsubject is a human.
 10. A method of treating a disease or condition in asubject in need thereof, which method comprises administering to saidsubject (a) a PDE3/PDE4 inhibitor which is9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-oneor a pharmaceutically acceptable acid addition salt thereofsimultaneously, separately or sequentially in combination with (b) aβ₂-adrenergic receptor agonist, wherein the disease or condition isselected from the group consisting of asthma, allergic asthma, hayfever, allergic rhinitis, bronchitis, emphysema, bronchiectasis, chronicobstructive pulmonary disease (COPD), adult respiratory distresssyndrome (ARDS), steroid resistant asthma, severe asthma and paediatricasthma.
 11. A method of treating a disease or condition in a subject inneed thereof, which method comprises administering to said subject (b) aβ₂-adrenergic receptor agonist simultaneously, separately orsequentially in combination with (a) a PDE3/PDE4 inhibitor which is9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-oneor a pharmaceutically acceptable acid addition salt thereof, wherein thedisease or condition is selected from the group consisting of asthma,allergic asthma, hay fever, allergic rhinitis, bronchitis, emphysema,bronchiectasis, chronic obstructive pulmonary disease (COPD), adultrespiratory distress syndrome (ARDS), steroid resistant asthma, severeasthma and paediatric asthma.